Cell surface molecule-induced macrophage activation

ABSTRACT

This invention provides cells containing recombinant polynucleotides coding for cell surface molecules that, when expressed in the cell, result in rejection of the cell by the host immune system. The invention also provides methods of using such cells, and capsules for delivery of biologically active molecules to a patient.

TECHNICAL FIELD OF THE INVENTION

This invention relates generally to the immunoglobulins, and moreparticularly to a cell surface molecule-induced macrophage activationthat results in the rejection by the host of the cell expressing thecell surface molecule.

BACKGROUND OF THE INVENTION

Cell therapy seeks to provide biologically active molecules to a patientby implanting cells that produce the biologically active molecules intothe patient. In unencapsulated cell therapy approaches, “naked” cellsare implanted. Several approaches have been taken to prevent rejectionof the implanted “naked” cells. Treatments include immunosuppression ofthe patient, preclearance from the recipient serum of naturalantibodies, or administration of high doses of low molecular weighthaptens to inhibit natural antibody binding to transplanted tissue.Alternatively, researchers have proposed the alteration of cells toreduce or eliminate expression of antigen or epitopes that stimulaterejection of the cells or tissue by natural antibodies in a recipient.However, using mitotically active cells creates a risk of tumorigenicityof the implanted tissue.

In encapsulated cell therapy approaches, a permselective physicalbarrier immunoisolates the implanted tissue from the host tissue. Thebarrier permits passage of the desired molecules between the patient andthe encapsulated tissue, but protects the encapsulated cells or tissuefrom destruction by the immune system of the patient. Use of xenogeneictissue or cells in encapsulated cell therapy also acts as a “safetyfeature,” because encapsulated cells are rejected by the patient'simmune system if the capsule breaks or ruptures.

A patient's immune system has several components, some of which areuseful for encapsulated cell therapy and some of which are undesirable.In one component, phagocytes scavenge target cells, such as thexenogeneic cells described above. In particular, antibody-dependentcell-mediated cytotoxicity (ADCC) has an important role in thedestruction of many target cells, including tumor cells, by macrophages.Opsonization of target cells with immunoglobulin G (IgG) enhances theremoval of these materials from a host. The role of macrophages in thedestruction of target cells by ADCC in the presence of specificantibodies has been well established. While the selectivity ofmacrophage targeting is based on antibody specificity, the lytic attackon the target cells is triggered by Fc receptor-mediated ADCC.

Another component of the immune system is the activation of thecomplement system. The two pathways of complement activation (theclassical and the alternative pathways) are both directed at a centralstep in complement activation, the cleavage of C3. A single terminalpathway is the formation of a membrane attack complex (MAC). Theclassical pathway is normally activated by antigen-antibody complexes.,where certain antibodies are complement fixing (capable of binding tocomplement to cause activation of the classical pathway). Activation ofthe classical pathway can be initiated with binding of C1q, the firstfactor of complement cascade, to the Fc region of immunoglobulin. Then,a cascade of proteolytic events results in the activation of C5convertase, which cleaves C5 into C5b and C5a. The C5b then binds C6,C7, C8 to form a C5b-8 complex. Binding of C9 molecules to C5b-8 formsC5b-9 (the MAC), which inserts into lipid bilayers and formstransmembrane channels that permit bidirectional flow of ions andmacromolecules. By this mechanism, complement causes lysis of the cells.

The complement system is important in host defense, but activation atinappropriate sites or to an excessive degree can cause host tissuedamage. Complement is a factor in the causation or propagation of tissueinjury in numerous diseases. For encapsulated cell therapy approaches inhumans, therefore, (1) unencapsulated cells should be rejectedimmediately by the host; (2) encapsulated cells should benon-immunogenic to the host; and (3) the cell elimination process shouldnot lead to immunological memory of the host. Accordingly, it would bedesirable to be able to deliver a biologically active molecule to humanpatients using encapsulated cells or tissue that both have a humanimmunological “background,” but also provide the safety feature ofrejection by the patient in the event of capsule rupture or failure.

SUMMARY OF THE INVENTION

The invention provides novel approaches for expressing naturally type Icell surface molecules (i.e., with the carboxy terminus [C-terminus]projecting toward the cytosol and the amino-terminus [N-terminus]projecting away from the cell surface) as type II molecules (i.e., withthe N-terminus projecting toward the cytosol and the C-terminusprojecting away from the cell surface). The biological function ismaintained in the type II orientation. Using this approach (1) cellsexpressing such molecules can be used as therapeutic agents and (2) ascreening process can evaluate the function of novel molecules that werenot previously available for testing. The invention also provides anovel approach to predetermining the fate of the transformed cells.Thus, the invention is new aspect of gene therapy and tumor therapy.While gene therapy is a new field, conferring cytotoxic sensitivity ontumor cells has been an area of active research. The strategy describedhere can be used to target tumor cells; tumor cells expressingimmunostimulatory cell surface polypeptides are therefore moresusceptible to macrophage clearance.

The invention provides novel immunostimulatory cell surfacepolypeptides, novel recombinant polynucleotides encodingimmunostimulatory cell surface polypeptides, and transformed cellscontaining the recombinant polynucleotides. When a transformed cellcontaining a recombinant polynucleotide expresses the encodedimmunostimulatory cell surface polypeptide in a host, the host undergoesan immune response that results in rejection of the transformed cell bythe host. The host immune response can include the activation ofphagocytes, such as macrophages, but does not include complementfixation. In a specific embodiment, the immunostimulatory cell surfacepolypeptides is a chimeric polypeptide containing the human transferrinreceptor membrane domain anchors a human IgG₁ Fc to the surface of thecell plasma membrane in a “reversed orientation,” thus mimicking theconfiguration of IgG during opsonization. The transformed cellscontaining the recombinant polynucleotides of the invention aretherapeutically useful for the treatment of many disorders.

The invention also provides diagnostic methods for identifying noveltherapeutics. In one embodiment, the invention is a method for testingphagocytes for response to an immunostimulatory cell surfacepolypeptide. A phagocyte is contacted in vitro with a transformed cellcontaining a recombinant polynucleotide. The recombinant polynucleotideis a promoter operably linked with a polynucleotide coding for animmunostimulatory cell surface polypeptide, and the immunostimulatorycell surface polypeptide activates phagocytes, but does not fixcomplement. The phagocytic activity of the phagocyte is compared withcontrol phagocyte; and increased phagocytic activity indicates that thephagocyte responds to the immunostimulatory cell surface polypeptide. Inanother embodiment, the invention is a method for identifying a compoundthat modulates phagocyte response to an immunostimulatory cell surfacepolypeptide. A phagocyte is contacted in vitro with a transformed cellcontaining a recombinant polynucleotide. The process is then repeated bycontacting a phagocyte in vitro with a test compound and the transformedcell containing a recombinant polynucleotide. The phagocytic activity ofthe phagocyte in the absence of the test compound is compared with thephagocytic activity of the phagocyte in the presence of the testcompound. A change in the phagocytic activity indicates that the testcompound modulates phagocyte response to the immunostimulatory cellsurface polypeptide.

The invention further provides a method for stimulating phagocyteactivity. A transformed cell containing a recombinant polynucleotidecontaining a promoter operably linked with a polynucleotide coding foran immunostimulatory cell surface polypeptide is administered to a host.In one embodiment, the stimulated phagocyte is a macrophage, especiallya macrophagic tumor cell. In another embodiment, the transformed cellcontains a therapeutic compound, such as an anti-tumor compound.

The invention provides a method for modulating an immune response in ahost. A transformed cell containing a recombinant polynucleotide with apromoter operably linked with a polynucleotide coding for animmunostimulatory cell surface polypeptide is administered to the host.The administration stimulates an immune response to the transformedcell, because the activation of phagocytes, especially macrophages, actsto regulate both T and B lymphocytes. Macrophages engulf the transformedcell and present the antigenic determinants from the transformed cell toT cells, stimulating an immune response. In one embodiment, the cellexpresses, on the cell surface, a “second antigen,” such that the hostproduces an immune response against the second antigen from thetransformed cell. The immunostimulatory cell surface polypeptideenhances the cellular interaction with macrophages. As a result of thisenhanced cellular interaction, the second antigen is presented as atarget for T-cells. In one embodiment, the transformed cell expressesthe second antigen from a recombinant polynucleotide.

The invention provides a method for ablating undesirable target cells,such as tumor cells in a patient, by the targeted delivery of therecombinant polynucleotides of the invention followed by eitherconstitutive or inducible expression of encoded polypeptide. Thedelivery of the immunostimulatory cell surface polypeptide of theinvention into solid tumors results in the selective phagocyte-mediatedablation of the undesirable cells.

The invention provides a method for the treatment of autoimmunedisorders in a host, by eliminating autoreactive T-cells. Transformedcells containing a recombinant polynucleotide comprising a promoteroperably linked with a polynucleotide coding for an immunostimulatorycell surface polypeptide are administered to a host with an autoimmunedisorder. The cells express a therapeutically effective amount ofimmunostimulatory cell surface polypeptide from the recombinantpolypeptide. The immunostimulatory cell surface polypeptide contactingmacrophages activates the macrophages to modulate host autoreactiveT-cells, thereby reducing the T-cell autoreactivity in the host.Macrophages specifically modulate Th1/Th2 responses. The reactiveness ofT-cells differ depending on the availability of co-stimulatory factors.Therefore, T-cells can be induced to become tolerant.

The invention provides a composition in which a transformed cell capableof expressing an immunostimulatory cell surface polypeptide isencapsulated in an immunoisolatory capsule. The transformed cells of theinvention are particularly useful when encapsulated for implantation ina human patient, because cells escaping from a ruptured capsule aredestroyed by the patient's immune system. A host immune response willnot be triggered by the transformed cells expressing animmunostimulatory cell surface polypeptide in an intact device. In caseof a device failure, however, the released cells are effectivelyeliminated by phagocytes without complement activation or the creationof an immune memory.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the rationale of the invention in one embodiment. FIG. 1Ashows the natural configuration of IgG opsonization. FIG. 1B shows anaturally occurring cell surface IgG anchored by type I transmembranedomain, with carboxyl terminus facing cytosol and amino terminus facingout side of cell. FIG. 1C shows a reverse cell surface Fc anchored bytype II transmembrane domain, with amino terminus facing cytosol andcarboxyl terminus facing out side of cell, mimicking IgG opsonization.

FIG. 2 shows the expression of reverse hTR-FcΔH (SEQ ID NO:4) in BHKcells, with the plasma membrane-associated hIgG determined by an ELISA.

FIG. 3 shows the effect of cell surface IgG Fc, either intact IgG orrecombinant reverse Fc, on superoxide production. FIG. 3A shows thedose-response effect of anti-serum opsonized BHK cells on superoxideproduction by mouse macrophages. FIG. 3B shows the effect of BHK-reverseFc clones on superoxide production by mouse macrophages. The results arepresented as V_(max) (nmoles O₂/10⁷ cells/min), with the numbers inparenthesis representing individual clones.

FIG. 4 shows the effect of anti-FCγRl mAb F(ab′)₂ on BHK-FcΔH-induced(SEQ ID NO:4) superoxide production in U937 cells. The solid barrepresents superoxide assay conditions without anti-FCγRl mAb treatment,and the shaded bar represents assay conditions with anti-FCγRl mAbpretreatment.

FIG. 5 shows the effect of BHK-FcΔH induced (SEQ ID NO:4) in complementactivation. C3a-desArg enzyme immunoassay was performed followingpretreatment of human serum with BHK-WT, BHK-FcΔH induced (SEQ ID NO:4)and immune complexes (IC).

DETAILED DESCRIPTION OF THE INVENTION Introduction

The invention provides novel recombinant polynucleotides encodingimmunostimulatory cell surface polypeptides that, when expressed by acell, result in rejection of that cell by the host immune system. Theinvention also provides transformed cells containing the recombinantpolynucleotides and methods for using the transformed cells. In specificembodiments described in EXAMPLE 1, a chimeric polypeptide containingthe human transferrin receptor membrane domain anchors a human IgG₁ Fcto the surface of the cell plasma membrane in a “reversed orientation”(SEQ ID NO:2 and SEQ ID NO:4), thus mimicking the configuration of IgGduring opsonization (FIGS. 1A and IC). The human IgG₁ chimericpolypeptide binds the Fc receptor (here, FcγRI) to activate phagocytes,such as macrophages, but avoids the undesirable characteristics of alsoactivating the complement cascade (“complement fixation”). A chronicallyactivated complement system can kill host cells, and accumulatingevidence suggests that this mechanism can cause many degenerativediseases, including inflammation and neurodegenerative diseases.

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description and from the claims.In the specification and the appended claims, the singular forms includeplural referents unless the context clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. All patents and publications citedin this specification are incorporated by reference.

Immunostimulatory cell surface polypeptide

An “immunostimulatory cell surface polypeptide” is a polypeptide,expressed on a cell surface, that is capable of stimulating an immuneresponse against the cell in a host. As used herein, a “biologicallyactive molecule” is an immunostimulatory cell surface polypeptide.Polypeptides appropriate for use as immunostimulatory cell surfacepolypeptides include the following:

a. opsonins such as IgG and C3b.

b. proteins with carbohydrate residues that interact with themannose-fucose receptor of phagocytes;

c. proteins capable of recognition by receptors on scavengermacrophages;

d. ligands for integrins; located on phagocytes;

e. glycoproteins, such as integrins and selectins; and

f. fucosyl transferase, which generates a Gal—Gal epitope recognized bymacrophages.

The immunostimulatory cell surface polypeptides can be used in either afull-length or a truncated form, as appropriate. In particular, theterms “region” and “domain” as used to describe an immunostimulatorycell surface polypeptide includes either a full-length immunostimulatorycell surface polypeptide or a part of the immunostimulatory cell surfacepolypeptide, such as the IgG regions and domains described below.

Immunoglobulin G (IgG) is the preferred immunostimulatory cell surfacepolypeptide for use in this invention. An IgG protein contains (1) anFab region (including the VH, VL and CH₁ domains); (2) a hinge region,and (3) an Fc region (including the CH₂ and CH₃ domains). The Fab regionis the region of an antibody protein which includes the antigen-bindingportions. The “hinge” region is a flexible area on the immunoglobulinpolypeptide that contains many residues of the amino acid proline and iswhere the Fc fragment joins one of the two Fab fragments. The Fc regionis the constant region on an immunoglobulin polypeptide; is located onthe immunoglobulin heavy chains; and is not involved in bindingantigens. The Fc region can bind to an Fc receptor on phagocytes. Theamino-proximal end of the CH₂ domain, especially amino acids 234 to 237,is important for binding of the Fc region to the Fc receptor. Fcreceptors, such as FcγRI, are integral membrane proteins located onphagocytic white blood cells, such as macrophages. The hinge region isimportant for regulating Fc—Fc receptor interactions, providingflexibility to the polypeptide and functioning as a spacer.

The immunoglobulin polynucleotide used for producing a recombinantimmunostimulatory cell surface polypeptide can be from any vertebrate,such as human or mouse (see, EXAMPLE 1). Preferably, the polynucleotideencodes an immunoglobulin having a substantial number of sequences thatare of the same origin as the host. For example, if a human is treatedwith a polypeptide of the invention, preferably the immunoglobulin is ofhuman origin. The immunoglobulin polynucleotide may code for a fulllength polypeptide or a fragment, such as a fragment of a larger fusionprotein, which includes an immunostimulatory cell surface polypeptideand a “second cell surface polypeptide.” Some advantages of using animmunoglobulin fusion protein include one or more of (1) possibleincreased avidity for multivalent ligands, (2) longer serum half-life,(3) the ability to activate effector cells by the Fc domain, and (4)ease of purification (for example, by protein A chromatography). EXAMPLE1 shows the construction and use of two immunoglobulin fusion proteins.

In one embodiment, IgG₁-Fc is expressed on the cell surface in a“reverse orientation” (see, FIG. 2). The Fc is in a reverse orientation(i.e., as a type II protein, with the N-terminus projecting toward thecytosol and the C-terminus projecting away from the cell surface) ascompared with the orientation of Fc in the naturally type I cell surfaceIgG polypeptide (with the C-terminus projecting toward the cytosol andthe N-terminus projecting away from the cell surface). In specificembodiments provided in EXAMPLE 1 (SEQ ID NO:2 and SEQ ID NO:4), thereverse orientation results from the fusion of the Fc region with a typeII membrane protein transmembrane domain. Cell surface Fc expressed inthe reverse orientation, a novel design in this invention, retains thebiological function of IgG₁ Fc of binding Fc receptor to mediatemacrophage activation, while simultaneously losing the complementfixation capability, as described in EXAMPLE 1.

Only when the CH₁ (e.g., SEQ ID NO:2) or CH₁ and hinge regions (e.g.,SEQ ID NO:4) are deleted from IgG₁ is a high level of plasma membraneexpression of reverse Fc achieved (see, FIG. 2). Full length IgG₁ heavychain constant region (hTR-Fc) resides primarily in the endoplasmicreticulum. Removal of the CH₁ domain allows translocation the chimerafrom the endoplasmic reticulum to the plasma membrane.

Phagocyte activation

Immunostimulatory cell surface polypeptides and their receptors areimportant for the clearance and destruction of foreign materials,including mammalian cells or bacteria. Immunostimulatory cell surfacepolypeptides and their receptors activate the phagocytosis and ADCC. Theprocess begins with opsonization of the foreign materials. An opsonin isan agent, usually an antibody or complement components, that makes acell or microbe more vulnerable to being engulfed by a phagocyte;opsonization is the process of coating a cell with opsonin. A phagocyteis an cell that engulfs and devours another; the process of engulfingand devouring is phagocytosis. Among the important phagocytes for thisinvention are macrophages and monocytes. Monocytes are a type of largewhite blood cell that travels in the blood but which can leave thebloodstream and enter tissue to differentiate into macrophages.Macrophages digest debris and foreign cells. Monocytes are generallycharacterized by the cell surface expression of CD14.

In a specific embodiment of the invention, cells coated withimmunoglobulins bind to phagocytes through the Fc receptors on thephagocytes. Phagocytes respond to signals from the Fc receptors byassembling cytoskeletal proteins, signaling cytoskeletal-proteinassembly by activation of protein tyrosine kinases, and by phagocytosingthe cell coated with immunoglobulin. IgG-FcγRI interaction activatesvarious biological functions such as phagocytosis, endocytosis, ADCC,release of inflammatory mediators and superoxide anion production.Macrophages possess organic anion transporter proteins that promote theafflux of anionic substances from the macrophage. Thus, FcγRI mediatesADCC by macrophages and triggers both phagocytosis and superoxideproduction. For that reason, the cells and methods of the inventionwhere the Fc domain of IgG is expressed on the surface of the cell tointeract with phagocyte Fc receptor cause phagocytes to bind to the cellexpressing the Fc domain of IgG, inducing ADCC. The IgG₁ and IgG₃isotypes, that interact with the high affinity receptor FcγRI onmacrophages, are preferred for the cells and methods of the invention.

Macrophages can also present antigens to T cells. In this way,macrophages are involved in other components of the immune response,including the humoral immune response (antibody production) and cellularimmune response.

Absence of complement fixation

A major function associated with human IgG₁ is the activation ofcomplement, an undesirable characteristic for encapsulated cell therapy.Activation of complement pathways may lead to a variety of undesirablebiologic actions, such as damage cells within a device. The transformedcells of the invention have the useful characteristics of being (1)cells expressing immunostimulatory cell surface polypeptide; (2) so thatthe immunostimulatory cell surface polypeptide activates macrophages;but (3) the immunostimulatory cell surface polypeptide does not activatethe complement cascade.

In EXAMPLE 1, in an embodiment where the immunostimulatory cell surfacepolypeptide is a Fc in a reversed orientation, a C3a enzyme immunoassayshowed that no complement fixation occurs (FIG. 5). By contrast, immunecomplexes containing intact IgG activated complement effectively. Thisis consistent with observations that IgG₁ heavy chain CH₁ and hingedomains are important for complement activation. Thus, although a highlevel of Fc is expressed on the cell surface, the chimera of IgG₁ heavychain with CH₁ and hinge deletion (e.g., SEQ ID NO:4) do not activatecomplement, despite being a potent stimulus for macrophages.

Immunostimulatory cell surface polypeptide fused to a second cellsurface polyveptide

In one embodiment, the immunostimulatory cell surface polypeptide isfused to a second cell surface polypeptide to form a single polypeptideexpressed at the cell surface. The second cell surface protein anchorsthe immunostimulatory cell surface polypeptide to the exterior of thecell. Examples of second cell surface proteins that may be suitable forsuch use include transferrin, CD10, CD13, CD23, CD26, CD38, CD71, CD72,CD74, 4F2, BP-1, endoglin, Ly-49, M-ASGP-BP, NKG2A, NKR-PI, and PC-1. Ofthese cell surface molecules, CD71, CD72, BP-1, endoglin, Ly-49, NKR-PIand PC-1 are preferred because these polypeptides are known to dimerizeand can facilitate dimerization of the Fc region of the antibody, forthe enhanced stability of Fc.

The second cell surface polypeptide can be human transferrin receptor(hTR), a type II cell surface protein. In a specific embodiment, theextracellular region of the transferrin receptor substitutes for thenative hinge region of IgG to anchor Fc (residues 89-97). The hTRfragment is approximately equal in length, but not in amino acididentity, to native IgG₁ hinge, and may effectively provide spacerfunctions similar to that of the hinge region. Also, the hinge region ofIgG provides intermolecular disulfide bonds between heavy and lightchains using critical cysteine residues. The hTR region (1-97) containsat least one cysteine (C89) to mimic the hinge region function, byallowing multimeric association of the hTR-FcΔH monomers.

Transformed cells and recombinant genetic techniques

A “transformed” cell is a cell or progeny of a cell into which has beenintroduced, by means of recombinant genetic techniques, a polynucleotideencoding a cell surface protein. The term “recombinant” refers to aproduct of human intervention. The transformed cell may be any humancell that can express an immunostimulatory cell surface polypeptide. Anysuitable source of human tissue, can be used as a source for generatingtransformed cells, including publicly available immortalized cell linesand dividing primary cell cultures. Examples of human cell lines includehuman neural stem or progenitor cells; RPMI 2650, HT-1080 or SW-13epithelial cells; HL-60 macrophage cells; CCRF-CEM or RPMI 8226 lymphoidcells; and WI-38, HEL1, MRC-5 or IMR-90 fibroblast cells. Useful humancell lines have the ability to be easily transfected, and to secreteproteins and peptides.

The transformed cells can be from other mammalian sources, for example,from rodents. In EXAMPLE 1, baby hamster kidney (BHK) cells opsonizedwith different concentrations of antibody stimulated a dose-dependentincrease in superoxide production (FIG. 3A). Similarly, transformedBHK-Fc cells induce superoxide production. Thus, the presence of IgG Fcon the cell surface of transformed hamster cells activate macrophages.

A recombinant polynucleotide encoding an immunostimulatory cell surfacepolynucleotide can be constructed in a standard DNA expression vectorand introduced to a cell for expression within the cell. Polynucleotidesfor insertion into cloning vectors, for example coding polynucleotides,can be constructed using the polymerase chain reaction (PCR) to amplifyappropriate polynucleotides. Polynucleotide synthesis and purificationtechniques are described in Sambrook et al. (Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1989) andCurrent Protocols in Molecular Biology (Ausubel et al., Eds., WileyInterscience, N.Y. 1993). The PCR procedure is performed by well-knownmethodology. See, for example, Ausubel et al, and Bangham (In Protocolsin Molecular Genetics, Humana Press, 1991). Moreover, PCR kits can bepurchased from companies such as Stratagene Cloning Systems (La Jolla,Calif.) and Invitrogen (San Diego, Calif.). The products of PCR aresubdloned into cloning vectors. The use of PCR for bacterial host cellsis described, for example, by Hofmann et al. (In PCR Protocols andApplications, White, Ed., Humana Press, 1993, pp. 205-210), and byCooper et al. (Id. at pp. 305-316). Coding polynucleotides areconstructed by PCR in EXAMPLE 1.

A “vector” is a replicon to which coding polynucleotide is attached, soas to bring about the replication or expression of the attached codingpolynucleotide . Vectors can be used for the transformation of cells ingene manipulation bearing a coding polynucleotide corresponding toappropriate polypeptides that, when combined with appropriate controlsequences, confer specific properties on the transformed cell.Recombinant vectors are constructed by cutting and joiningpolynucleotides from different sources using restriction enzymes andligases.

Vectors include cloning vectors and expression vectors. A cloning vectoris a polynucleotide, such as a plasmid, cosmid or bacteriophage, thatcan replicate autonomously in a host prokaryotic or eukaryotic cell.Cloning vectors typically contain one or a small number of restrictionendonuclease recognition sites at which polynucleotide sequences can beinserted in a determinable fashion without loss of an essentialbiological function of the vector, as well as a marker gene that issuitable for use in the identification and selection of cellstransformed with the cloning vector. Suitable cloning vectors aredescribed by Sambrook et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1989); Current Protocols inMolecular Biology (Ausubel et al., Eds., Wiley Interscience, N.Y. 1993),and Molecular Biology LabFax (Brown, Ed., Academic Press, 1991). Cloningvectors can be obtained, for example, from GIBCO/BRL (Gaithersburg,Md.), Clontech Laboratories, Inc. (Palo Alto, Calif.), PromegaCorporation (Madison, Wis.), Stratagene Cloning Systems (La Jolla,Calif.), Invitrogen (San Diego, Calif.), and the American Type CultureCollection (Rockville, Md.).

Cloned variants are amplified by transforming competent bacterial cellswith a cloning vector and growing the bacterial host cells in thepresence of the appropriate antibiotic (see, for example, Sambrook etal., and Ausubel et al., above). Bacterial host cells are then screenedfor the appropriate clones.

The resulting recombinant polynucleotide or relevant parts can be clonedfrom cloning vectors into expression vectors, which expression vectorshave characteristics permitting higher levels of, or more efficientexpression of, the resident polynucleotides. These constructs mayrequire a promoter that initiates transcription of the inserted codingpolynucleotide. A “promoter” is a polynucleotide sufficient to directtranscription, including those promoter elements which are sufficient torender promoter-dependent gene expression inducible. Typically, apolynucleotide encoding a biologically active cell surface proteinpolypeptide is operably linked to a promoter. “Operably linked” refersto a juxtaposition where the components are configured so as to performtheir usual function. Thus, promoter operably linked to a codingpolynucleotide is capable of effecting the expression of the codingpolynucleotide. By “operably linked” is meant that a coding polypeptideand a promoter are functionally connected to permit gene expression whenthe appropriate factors (e.g., transcriptional activator proteins) arebound to the regulatory sequence. The orientation or placement of theelements of the vector is not strict, so long as the operable linkagerequirement is fulfilled for control of and expression of the codingpolynucleotide. A “mammalian” promoter is a polynucleotide that directstranscription in a mammalian cell (e.g., a promoter of a mammal or avirus that infects a mammal). Transcriptional regulatory sequencesinclude a promoter region sufficient to direct the initiation of RNAsynthesis. Suitable eukaryotic promoters include the promoter of themouse metallothionein I gene (Hamer et al., J Molec. Appl. Genet. 1:273, 1982); the TK promoter of Herpes virus (McKnight, Cell 31: 355,1982); the SV40 early promoter (Benoist et al., Nature 290: 304, 1981);the Rous sarcoma virus promoter (Gorman et al., Proc. Nat'l Acad. Sci.USA 79: 6777 1982); and the cytomegalovirus promoter (Foecking et al.,Gene 45: 101, 1980).

Many genetic constructs and methods for expressing heterologous genes incells of mammals are known in the art and are suitable for use in theinvention. For example, expression of a cell surface protein can beaccomplished with conventional gene therapy methods, such as those thatemploy viral vectors (e.g., vectors derived from retroviruses,adenoviruses, herpes viruses, vaccinia viruses, polio viruses, sindbisviruses, or adeno-associated viruses).

Constitutive expression

In one embodiment, the immunostimulatory cell surface polypeptide isconstitutively expressed in the transformed cell. Constitutiveexpression is achieved by the use of a vector with a constitutivepromoter. For example, the vector pRc/CMV (Invitrogen, San Diego,Calif.) provides a high level of constitutive transcription frommammalian enhancer-promoter sequences. Another constitutive promoter isthe interferon-inducibleMx-1 promoter. The level of expression maydepend on the immunostimulatory cell surface polypeptide used, on thevector copy number, or the vector cellular or genomic location, by doesnot, by contrast with inducible expression, depend on the addition offactors.

Constitutive expression can occur when the recombinant polynucleotidebecomes part of the genome of an organism (i.e., either stablyintegrated or as a stable extrachromosomal element) that develops fromthat cell. Such a polynucleotide may include a gene which is partly orentirely heterologous (i.e., foreign) to the transgenic organism, or mayrepresent a gene homologous to an endogenous gene of the organism.

Increased constitutive or inducible expression can be achieved byincreasing or amplifying the vector copy number using amplificationmethods well known in the art. Such amplification methods include, e.g.,DHFR amplification (see, e.g., Kaufinan et al., U.S. Pat. No. 4,470,461)or glutamine synthetase (“GS”) amplification (see, e.g., U.S. Pat. No.5,122,464, and European Patent Application Publication No. EP 338,841).Expression vectors containing the geneticin (G418) or hygromycin drugselection genes (Southern, In Vitro, 18: 315, 1981, Southern & Berg, JMol. Appl. Genet., 1: 327, 1982) are also useful. These vectors canemploy a variety of different enhancer/promoter regions to drive theexpression of both a biologic gene of interest and a gene conferringresistance to selection with toxin such as G418 or hygromycin B. TheG418 resistance gene codes for aminoglycoside phosphotransferase (APH)which enzymatically inactivates G418 added to the culture medium. Onlythose cells expressing the APH gene will survive drug selection usuallyresulting in the expression of the second biologic gene as well. Thehygromycin B phosphotransferase (HBH) gene codes for an enzyme thatspecifically modifies hygromycin toxin and inactivates it. Genesco-transfected with or contained on the same plasmid as the hygromycin Bphosphotransferase gene will be preferentially expressed in the presenceof hygromycin B.

Inducible expression

In another embodiment, the expression vector encoding theimmunostimulatory cell surface polypeptide is inducible. High levels ofexpression can be accomplished by the addition of a regulatory regionwhich provides increased transcription of downstream sequences in theappropriate host cell. For a mammalian host, the transcriptional andtranslational regulatory signals preferably are derived from viralsources, such as adenovirus, bovine papilloma virus, simian virus, orthe like, in which the regulatory signals are associated with aparticular gene which has a high level of expression. Suitabletranscriptional and translational regulatory sequences also can beobtained from mammalian genes, such as actin, collagen, myosin, andmetallothionein genes.

The invention therefore provides a “suicide gene” for use in therapy. Inthis embodiment, transformed cells are used to provide apharmacologically effective treatment. When treatment is no longerdesirable (for any reason, such as the treatment being unsuccessful orthe treatment being successfully completed), expression of theimmunostimulatory cell surface polypeptide is induced. The expressionresults in the transformed cell being effectively removed from thepatient.

Expression in the central nervous system

The brain is an immunologically privileged site, sheltered fromcirculating cells and proteins of the immune system; but a growing bodyof evidence implicates complement in numerous brain diseases (see reviewby Morgan et al., Immunopharmacology 38(1-2):43-50, 1997). Complementsynthesis and activation in the brain are important in immune defense atthis site, but may also be of importance in CNS conditions such asAlzheimer's disease, ischaemia and Parkinson's disease, as well as inperipheral disorders such as myocardial ischaemia andxenotransplantation (McGeer & McGeer, Drugs55(6):739-46, 1998). InAlzheimer disease (AD) cases, positive staining for classical pathwaycomplement proteins C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 and C9 wasobserved in pyramidal neurons and senile plaques (Terai et al., BrainRes 769(2):385-90, 1997; Shen et al, Brain Res 769(2):391-5, 1997). Somebrain cells synthesize complement and also express specific receptors;some are exquisitely sensitive to the lytic effects of complement.Complement activation causes neuronal cell death in vitro, and thisneurodegenerative process is regulated by homologous restriction, asdescribed by Shen et al. (Brain Res Brain Res Protoc 1(2):186-94, 1997).Thus, the cells, compositions, and methods of the invention are usefulin modulation of the central nervous system immune response.

In one embodiment, the promoter can be cell-specific, tissue-specific,or stage-specific, to express the immunostimulatory cell surfacepolypeptide in neural cells with increased specificity. Examples ofexpression vectors that can be employed are the commercially availablepRC/CMV, pRC/RSV, and pCDNAINEO (Invitrogen), where the viral promoterregions of interest are replaced with promoter sequences that are notsubject to the down regulation experienced by viral promoters within thecentral nervous system. For example, the GFAP promoter can be employedfor the transfection of astrocytes and the MBP promoter can be used inoligodendrocytes. Other promoters include, but are not limited to, thepromoters of hDBH (human dopamine beta hydroxylase) (Mercer et al.,Neuron 7: 703-716, 1991)), hTH (human tyrosine hydroxylase) (Kaneda etal, Neuron 6: 583-594, 1991), hPNMT (human phenylethanolamineN-methyltransferase) (Baetge et al., PNAS, 85: 3648-3652, 1988), mGFAP(mouse glial fibrillary acidic protein) (Besnard et al., J Biol. Chem.,266: 18877-18883, 1991)), myelin basic protein (MBP), mNF-L (mouseneurofilament-light subunit) (Nakahira et al., J Biol. Chem., 265:19786-19791, 1990), hPo (human P_(o), the promoter for the gene encodingthe major myelin glycoprotein in the peripheral nervous system) (Lemkeet al., Neuron, 1: 73-83, 1988), mMT, rNSE (rat neuron-specific enolase)(Sakimura, et al, Gene 60: 103-113, 1987), and the like.

Diagnostic methods

The transformed cells or immunostimulatory cell surface polypeptides ofthe invention are diagnostically useful for the detection of macrophageresponse to immunostimulatory cell surface polypeptides, for example, ininflammation. Biological samples, e.g. blood or derivatives thereof,biopsies, synovial fluid, etc., can be assayed. Assays may be performedon cell lysates, intact cells, frozen sections, etc. Many clinicallysignificant disorders are accompanied by inflammation, e.g. arthritis,bacterial infections, hypersensitivity, wound healing, etc. In arepresentative screening assay, the activation of in vitro phagocytes,such as macrophages, by in vitro cells expressing immunostimulatory cellsurface polypeptides is measured, as an in vitro test of a patient'smacrophage ability to ingest and kill specific target cells.Alternatively, purified or semi-purified immunostimulatory cell surfacepolypeptide may be bound to an insoluble substrate, and used in lieu ofthe cells or tissue.

The immunostimulatory cell surface polypeptides are also diagnosticallyuseful in screening assays to determine whether an agent is effective ininterfering with the interaction between phagocytes andimmunostimulatory cell surface polypeptides. In a representativescreening assay, the activation of in vitro phagocytes, such asmacrophages, by in vitro cells expressing immunostimulatory cell surfacepolypeptides is measured. Agents, particularly peptides, aptamers,carbohydrates, small organic molecules, etc. are added to the mixture ofantibody and cells, and a measured reduction in phagocyte activityindicates that the compound reacts with the immunostimulatory cellsurface polypeptide.

The term “agent” describes any molecule, e.g. protein or pharmaceutical,with the capability of altering or mimicking the physiological functionof a phagocyte. Generally a plurality of assay mixtures are run inparallel with different agent concentrations to obtain a differentialresponse to the various concentrations. Typically, one of theseconcentrations serves as a negative control, i.e. at zero concentrationor below the level of detection.

Also included in the screening method of the invention are combinatorialchemistry methods for identifying chemical compounds. See, for example,Plunkett & Ellman, (“Combinatorial Chemistry and New Drugs.” ScientificAmerican, April, 69, 1997). Areas of investigation for combinatorialchemistry are the development of therapeutic treatments. Drug screeningidentifies agents that provide a replacement, enhancement or regulationof function in affected cells. Of particular interest are screeningassays for agents that have a low toxicity for human cells.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons (Da).Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including, but not limited to: peptides, saccharides, fattyacids, steroids, purines, pyrimidines, derivatives, structural analogsor combinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification or amidification to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein—proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors or anti-microbial agents may be used. The mixture ofcomponents are added in any order that provides for the requisitebinding. Incubations are performed at any suitable temperature,typically between 4 and 40° C. Incubation periods are selected foroptimum activity, but may also be optimized to facilitate rapidhigh-throughput screening. Typically between 0.1 and 1 hours will besufficient.

Second antigen presentation

The invention provides a method for antigen presentation. Mildly orminimally antigenic substances (“second antigens”) are introduced to theimmune system. In one embodiment, the second antigens are on the surfaceof a transformed cell containing a recombinant polynucleotide with apromoter operably linked to a polynucleotide coding for animmunostimulatory cell surface polypeptide. In another embodiment, thetransformed cell containing a recombinant polynucleotide with a promoteroperably linked to a polynucleotide coding for an immunostimulatory cellsurface polypeptide actually makes the second antigenic substances. Forexample, the second antigen could be encoded by a recombinantpolynucleotide that has been used to transform the cell. In other words,transformed cells can be constructed by recombinant techniques toexpress the second antigen against which a strong immune response isdesired. This method of the invention is useful for promoting asignificant immune response against otherwise weak antigens. Among theweak antigens are tumor associated antigens or certain viral antigens,e.g., specific antigens of HIV-1, etc.

Method for stimulating phaeocyte activity

The invention provides a method for stimulating phagocyte activity. Atransformed cell containing a recombinant polynucleotide with a promoteroperably linked to a polynucleotide coding for an immunostimulatory cellsurface polypeptide is made to contact a phagocyte, such as amacrophage. The contact stimulates an increased phagocytic activity bythe phagocyte. The phagocyte may engulf the transformed cell. Thephagocyte may also or alternatively engulf other cells, or exhibitmeasurable properties of activated macrophages, such as those describedin EXAMPLE 1.

This method is useful for targeting a therapeutic compound to amacrophagic tumor. A therapeutic compound, for example an anti-tumorcompound, is introduced into a transformed cell containing a recombinantpolynucleotide with a promoter operably linked to a polynucleotidecoding for an immunostimulatory cell surface polypeptide. Thistransformed cell is introduced into a host, such as a patient. Thetransformed cell is targeted to the macrophagic tumor, whichphagocytoses the transformed cell. Thus, the therapeutic compound isdelivered.

Modulating an immune response

The invention also provides a method for modulating an immune responseusing transformed cells expressing immunostimulatory cell surfacepolypeptides. The term “modulate” means that the phagocyte activity iscontrolled or regulated in vivo by the methods of the present invention.The term “modulate” can mean either stimulating or inhibiting theresponse, depending on the situation. The method of the inventionincludes treatment of conditions in which either the immune reactionsare deleterious and suppression of such responses or immune reactions isdesirable, or conditions in which immune reactions are important andstimulation of such responses is desirable. An immunostimulatory cellsurface polypeptide may be useful in recruiting or activatingmacrophages that would enhance the immune response to a vaccine,stimulate a response for tumor rejection, or alter the response in aqualitative manner. Similarly, the immunostimulatory cell surfacepolypeptide may inhibit or depress an immune or inflammatory responsewhere desirable, such as in graft rejection responses after organ andtissue transplantations, or autoimmune disease. Some of the commonlyperformed transplantation surgeries include organs and tissues such askidneys, hearts, livers, skin, pancreatic islets and bone marrow.

Autoimmune disorders

“Autoimmune disorders” include the group of diseases caused by reactionsof the immune system to self antigens leading to tissue destruction. Theimmune system's response may be primarily humoral (autoantibodyproduction), primarily cellular (delayed-type hypersensitivity T-cellsand perhaps cytotoxic T-cells, i.e., “autoreactive T cells”), or, bothhumoral and cellular reactions may be induced. The highly specificreactivity of autoreactive T-cells is directed against externalcell-surface structures, internal cytoplasmic or nuclear constituents,or against secreted products produced by cells in different organs.There is clearly a problem of some kind regarding the development ofself-antigen reactive T_(H)-cells. In the method of the invention,expression of an immunostimulatory cell surface polypeptide results onthe elimination of autoreactive t-cells, thus reducing a factor involvedin the autoimmune disorder.

Some important autoimmune diseases include diabetes; autoimmunethyroiditis; multiple sclerosis and related demyelinating diseases;rheumatoid arthritis; systemic lupus erythematosis; and myastheniagravis. Other autoimmune and related disorders include, e.g.,polyarteritis nodosa: polymyositis and dermatomyositis: progressivesystemic sclerosis (diffuse scleroderma): glomerulonephritis: Sjogren'ssyndrome: Hashimoto's disease and Graves' disease: adrenalitis:hypoparathyroidism: pernicious anemia; uveitis pemphigus and pemphigoid;cirrhosis and other diseases of the liver; ulcerative coliris;myocarditis; regional enteritis; adult respiratory distress syndrome;local manifestations of drug reactions (dermatitis, etc.);inflammation-associated or allergic reaction patterns of the skin;atopic dermatitis and infantile eczema; contact dermatitis, psoriasislichen planus; allergic enteropathies; atopic diseases, e.g. allergicrhinitis and bronchial asthma; transplant rejection (heart, kidney,lung, liver, pancreatic islet cell, others); hypersensitivity ordestructive responses to infectious agents; poststreptococcal diseasese.g. cardiac manifestations of rheumatic fever, etc.

Encapsulation

The invention provides a composition in which transformed cellscontaining polynucleotides encoding an immunostimulatory cell surfacepolypeptide is encapsulated in an immunoisolatory capsule. An“immunoisolatory capsule” means that the capsule upon implantation intoa host minimizes the deleterious effects of the host's immune system onthe cells within the core. In the rare event that encapsulated cellsshould escape from a capsule whose integrity has been breached, thecells can be immediately eliminated by the host without triggeringspecific immunological memory. When encapsulated, the transformed celldoes not activate macrophages, but unencapsulated cells are effectivelyeliminated by the host.

Encapsulated cell therapy is a valuable therapeutic method. Encapsulatedcell therapy is based on the concept of isolating cells from a host'simmune system by surrounding the cells with a semipermeablebiocompatible material before implantation within the host. Usingencapsulation techniques, cells can be transplanted into a host withoutimmune rejection or use of immunosuppressive drugs. Useful biocompatiblepolymer capsules usually contain (a) a core which contains a cell orcells, either suspended in a liquid medium or immobilized within animmobilizing matrix, and (b) a surrounding or peripheral region ofpermselective matrix or membrane (“jacket”) which does not containisolated cells, which is biocompatible, and which is sufficient toprotect isolated cells if present in the core from detrimentalimmunological attack. Encapsulation hinders elements of the immunesystem from entering the capsule, thereby protecting the encapsulatedcells from immune destruction. The semipermeable nature of the capsulemembrane also permits the biologically active molecule of interest toeasily diffuse from the capsule into the surrounding host tissue. Thistechnique prevents the inherent risk of tumor formation and allows theuse of the transformed cells without immunosuppression of the recipient.Moreover, the implant may be retrieved if necessary or desired.

The capsule is made from a biocompatible material. A “biocompatiblematerial” is a material that, after implantation in a host, does notelicit a detrimental host response sufficient to result in the rejectionof the capsule or to render it inoperable, for example throughdegradation. The biocompatible material is relatively impermeable tolarge molecules, such as components of the host's immune system, but ispermeable to small molecules, such as insulin, growth factors,nutrients, while metabolic waste to be removed. A variety ofbiocompatible materials are suitable for delivery of growth factors bythe composition of the invention. Numerous biocompatible materials areknown, having various outer surface morphologies and other mechanicaland structural characteristics. Preferably the capsule of this inventionwill be similar to those described by Aebischer et al. (InternationalPatent Application No. WO 92/19195); Baetge (International PatentApplication Publication No. WO 95/05452); or U.S. Pat. Nos. 5,639,275;5,653,975; 4,892,538; 5,156,844; 5,283,187; and 5,550,050. Such capsuleswill allow for the passage of metabolites, nutrients and therapeuticsubstances while minimizing the detrimental effects of the host immunesystem. Components of the biocompatible material may include asurrounding semipermeable membrane and the internal cell-supportingscaffolding. preferably, the transformed cells are seeded onto thescaffolding, which is encapsulated by the pemiselective membrane. Thefilamentous cell-supporting scaffold may be made from any biocompatiblematerial selected from the group consisting of acrylic, polyester,polyethylene, polypropylene polyacetonitrile, polyethyleneteraphthalate, nylon, polyamides, polyurethanes, polybutester, silk,cotton, chitin, carbon, or biocompatible metals. Also, bonded fiberstructures can be used for cell implantation (U.S. Pat. No. 5,512,600).Further, biodegradable polymers can be use as scaffolds for hepatocytesand pancreatic cells. as reviewed by Cima et al. (Biotech. Bioeng. 38:145-58, 1991). Biodegradable polymers include those comprised ofpoly(lactic acid) PLA, poly(lactic-coglycolic acid) PLGA, andpoly(glycolic acid) PGA and their equivalents. Foam scaffolds have beenused to provide surfaces onto which transplanted cells may adhere. Wovenmesh tubes have been used as vascular grafts. Additionally, the core canbe composed of an immobilizing matrix formed from a hydrogel, whichstabilizes the position of the cells. A hydrogel is a three dimensionalnetwork of cross-linked hydrophilic polymers in the form of a gel,substantially composed of water.

Various polymers and polymer blends can be used to manufacture thesurrounding semipermeable membrane, including polyacrylates (includingacrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers,polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulosenitrates, polysulfones (including polyether sulfones), polyphosphazenes,polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well asderivatives, copolymers and mixtures thereof. Preferably, thesurrounding semipermeable membrane is a biocompatible semipermeablehollow fiber membrane. Such membranes, and methods of making them aredisclosed by Aebischer & Wahlberg (U.S. Pat. No. 5,284,761; U.S. Pat.No. 5,158,881). The surrounding semipermeable membrane is formed from apolyether sulfone hollow fiber, such as those described by Wechs (U.S.Pat. No. 4,976,859) and Muller & Wechs (U.S. Pat. No. 4,968,733). Analternate surrounding semipermeable membrane material ispoly(acrylonitrile/covinly chloride).

The capsule can be any configuration appropriate for maintainingbiological activity and providing access for delivery of the product orfunction, including for example, cylindrical, rectangular, disk-shaped,patch-shaped, ovoid, stellate, or spherical. Moreover, the capsule canbe coiled or wrapped into a mesh-like or nested structure. If thecapsule is to be retrieved after it is implanted, configurations whichtend to lead to migration of the capsules from the site of implantation,such as spherical capsules small enough to travel in the recipient'sblood vessels, are not preferred. Certain shapes, such as rectangles,patches, disks, cylinders, and flat sheets offer greater structuralintegrity and are preferable where retrieval is desired.

When macrocapsules are used, preferably between 10³ and 10⁸ cells areencapsulated, most preferably 10⁵ to 10⁷ cells are encapsulated in eachdevice. Dosage may be controlled by implanting a fewer or greater numberof capsules, preferably between 1 and 10 capsules per patient.

The scaffolding may be coated with extracellular matrix (ECM) molecules.Suitable examples of ECM molecules include, for example, collagen,laminin, and fibronectin. The surface of the scaffolding may also bemodified by treating with plasma irradiation to impart charge to enhanceadhesion of cells.

Any suitable method of sealing the capsules may be used, including theemployment of polymer adhesives and/or crimping, knotting and heatsealing. In addition, any suitable “dry” sealing method can also beused, as described, e.g., in U.S. Pat. No. 5,653,687.

Many implantation sites are contemplated for the devices and methods ofthis invention. These implantation sites include the central nervoussystem, including the brain, spinal cord, and aqueous and vitreoushumors of the eye.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. These examples should in noway be construed as limiting the scope of the invention, as defined bythe appended claims.

EXAMPLE 1 CLONING AND EXPRESSION OF IgG₁ cDNA IN REVERSE ORIENTATION

In this EXAMPLE, IgG₁ Fc chimera, with the Fc in a reversed orientation,were constructed and successfully expressed on a cell surface. The Fcreceptor binding property was retained in the molecules, while thecomplement activation capability was absent.

Rationale of the design and construction of chimeric hTR-hIgG₁expression cassette and chimeric hTR-mIgG₁ expression cassette

The rationale of this EXAMPLE was to genetically modify cells to expressIgG Fc in a reversed orientation on the cell surface, mimicking naturalIgG opsonization for cell elimination. The concept is presented in FIG.1. In the case of IgG opsonization, IgG binds to the target through Fabportion of the molecule, exposing the Fc portion for Fc receptor binding(FIG. 1A). To express Fc in a similar configuration, the Fc moleculemust be anchored to the plasma membrane in a reverse orientation withrespect to naturally occurring membrane bound IgG (FIG. 1B). Such areversed Fc must be anchored by the N-terminus with the carboxylterminus projecting away from the cell surface (FIG. 1C), in contrast tothe naturally occurring membrane bound IgG, which is anchored to themembrane via the Fc portion (FIG. 1B). A type II transmembrane domain isnecessary to achieve such a configuration. The hTR is a type IItransmembrane protein with the transmembrane region also functioning asa signal peptide. The transmembrane domain of the human transferrinreceptor was fused in-frame to the N-terminus of the second and thirddomains of human immunoglobulin G1 heavy chain constant region. Thisfusion molecule was designed to take advantage of the type II membraneanchor property of the transferrin receptor to express the Fc portion ofthe molecule in a reverse orientation, such that the Fc portionprojected away from the cell surface. This is in contrast to theconventional cell surface IgG₁ which is anchored by a C-terminal type Itransmembrane domain.

Taking advantage of such a property, a chimeric protein containing thehTR transmembrane domain and human IgG Fc domain was designed. The cellsurface expressed reverse Fc no longer activated complement but retainedFc receptor binding capability, and activated superoxide production bymacrophages. This activity was completely blocked by a FcγRI-specificmonoclonal antibody.

Primers

To facilitate the cloning of IgG₁ RT-PCR products into the pcDNA3.1(−)expression vector, synthetic restriction enzyme sites were engineered atthe 5′ ends of the above oligonucleotides. Specifically, BamHI and EcoRIsites were engineered in oligonucleotides #400 (SEQ ID NO:5) and #404,respectively, as underlined below. Oligonucleotides #401 (SEQ ID NO:6)and #405 contain the synthetic HindIII restriction sites at the 5′ end,as double-underlined below.

For human IgG₁ RT-PCR, oligonucleotides #400 (SEQ ID NO:5) and #401 (SEQID NO:6) were used:

#400: 5′-CCC GGA TCC GCC TCC ACC AAG GGC CCA TGC GTC-3′

#401: 5′-CCC AAG CTT CAT TTA CCC GGA GAC AGG GAG AGG-3′

To generate the chimeric human transferrin receptor-human IgG₁(“hTR-hIgG₁”) fusion expression cassette, oligonucleotides #402 (SEQ IDNO:7) and #403 (SEQ ID NO:8) were used. Oligonucleotide #402 (SEQ IDNO:7) is specific for the human transferrin receptor and contains thesynthetic BamHI restriction site, as single underlined below:

#402: 5′-CCC GGA TCC GCC ACC ATG.ATG GAT CAA GCT AG-3′

Oligonucleotide #403 (SEQ ID NO:8) is specific for the human transferrinreceptor (GAC CGA TGG GCC CTT GGT GGA GGC) and the human IgG₁ (CTC AGTTTT TGG).

#403: 5′-GAC CGA TGG GCC CTT GGT GGA GGC CTC AGT TTT TGG-3′

For the mouse IgG₁ RT-PCR, oligonucleotides #404 (SEQ ID NO:9) and #405(SEQ ID NO:10) were used):

#404: 5′-CCC GAA TTC GCC AAA ACG ACA CCC CCA TCT G-3′

#405: 5′-CCC AAG CTT CAT TTA CCA GGA GAG TGG GAG AG-3′

To generate the chimeric human transferrin receptor-mouse IgG₁(“hTR-mIgG₁”) fusion expression cassette, oligonucleotides #406 (SEQ IDNO:11), #407 (SEQ ID NO:12), #408 (SEQ ID NO:13), and #409 (SEQ IDNO:14) were used. Oligonucleotides #407 (SEQ ID NO:12) and #408 (SEQ IDNO:13) are complementary to each other:

#407: 5′-TGT CCT TTT GGC CTC AGT TTT TGG TTC TAC-3′

#408: 5′-CCA AAA ACT GAG GCC AAA ACG ACA CCC CCA-3′

Oligonucleotide #408 has the 5′ 12 nucleotides identical to the hTRsequence and the 3′ 18 nucleotides identical to the mIgG₁ sequence.Oligonucleotide #407 (SEQ ID NO:12) has the 5′ 12 nucleotides identicalto the mIgG₁ sequence and the 3′ 18 nucleotides identical to the hTRsequence.

Oligonucleotides #406 (SEQ ID NO:11) and #409 (SEQ ID NO:14) arespecific for the hTR and MIgG₁ sequences, respectively. As underlinedbelow, oligonucleotide #406 (SEQ ID NO:11) contains a synthetic EcoRIsite at the 5′ end, while oligonucleotide #409 (SEQ ID NO:14) containsan internal BamHI restriction site.

#406: 5′-CCC GAA TTC GCC ACC ATG ATG GAT CAA GCT AG-3′

#409: 5′-GTG TGC ACA CCG CTG GAC AGG GAT CCA GAG-3′

PCR reactions

N-terminal deletions of the Fc portion of human IgG₁, lacking the Fab,CH₁ or the hinge domains, were created by the polymerase chain reaction(PCR) and fused in frame to the transmembrane domain of the humantransferrin receptor (hTR). Residues 1-97 of hTR, which includes theputative signal sequence and transmembrane domain, and human IgG₁N-terminal deletion fragments were amplified from human spleen cDNA(Clontech, San Diego, Calif.) by PCR using the Advantage GC Genomic PCRkit (Clontech, San Diego, Calif.). The IgG₁ region encoding the CH₁,hinge, CH₂, and CH₃ domains was generated by RT-PCR of the human andmouse spleen total RNA. Total RNA was extracted from human or mousecells by the acid/phenol method described by Chomczynski & Sacchi (Anal.Biochem., 162: 156-159, 1987). RT-PCR was performed as previouslydescribed by Gandelman et al (J Neurochem., 56: 1024-1029, 1990). Thesource of the human transferrin receptor (“hTR”) was the plasmid HBMAC38(ATCC 100808) from the American Type Culture Collection (“ATCC”) inRockville, Md.

Briefly, 0.5 μg of total RNA was reverse transcribed to generate cDNA ina 20 ml reaction mixture according to Krug & Berger (Methods Enzymol.,152: 316-325, 1987). One to 5 μl of each reaction mixture was added tomake a final 50 μl PCR mixture containing 10 pmol each ofoligonucleotides #400 (SEQ ID NO:5) and #401 (SEQ ID NO:6) for the humanIgG₁ RT-PCR, and #404 (SEQ ID NO:9) and #405 (SEQ ID NO:10) for themouse IgG₁ RT-PCR. One hundred ng of template DNA HBMAC38 was added to a50 μl PCR reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mMKCl, 800 nM of each of four dNTP, 2 mM MgCl₂, 400 nM each ofoligonucleotides #402 and #403, and 2.5 units of Taq DNA polymerase(Boehringer Mannheim, Germany). PCR reactions were also carried outusing oligonucleotide pairs #406 (SEQ ID NO:11) and #407 (SEQ ID NO:12);and oligonucleotide pairs #408 (SEQ ID NO:13) and #409 (SEQ ID NO:14);on templates HBMAC38 and pcDNA3.1(−)-mIgG₁ plasmids, respectively.Reaction mixtures were subjected to 30 cycles of PCR. Each cycleconsisted of denaturation at 94° C. for 1 minute, annealing at 50° C.for 1 minute, and extension at 72° C. for 1 minute. The PCR fragment waspurified away from the used deoxynucleotides and salt in PCR reactionmixtures by the QIAquick solution purification kit (Qiagen, Chatsworth,Calif.) according to the manufacturer's protocol.

The purified human and mouse IgG₁ RT-PCR fragments were digested byBamHI/HindiII and EcoRV/HindIII, respectively, and subsequently ligatedinto pcDNA3.1(−)/BamHI/HindIII and pcDNA3.1(−)/EcoRI/HindIII,respectively, which was dephosphorylated by alkaline phosphatasetreatment. The hTR and IgG heavy chain fragments were ligated,generating the coding sequence for the chimeric molecule. These chimeraswere designated hTR-Fc (containing IgG₁ heavy chain constant region,full length), hTR-FcΔCH₁ (CH₁ domain deleted IgG₁ heavy chain constantregion; SEQ ID NO:1) and hTR-FcΔH (CH₁ and hinge deleted IgG₁ heavychain constant region; SEQ ID NO:3) respectively. The ligation mixtureswere transformed into DH5a, and ampicillin-resistant colonies werescreened for positive clones.

A cracking gel procedure (Promega Protocols and Applications Guide,19991) was used to screen out the positive clones. The identity of thecorrect clones was further verified by BamHI/DraIII double digestion.The plasmid DNA obtained from the ampicillin-resistant colonies weredigested by BamHI/HindIII and EcoRI/HindIII restriction endonucleases toverify the presence of human and mouse IgG₁ RT-PCR fragments,respectively. The resulting pcDNA3.1(−)-based human and mouse IgG₁intermediate cloning vectors were named pcDNA3.1(−)-hIgG₁ and pcDNA3.1(−)-mIgG₁, respectively.

Characterization and localization of the fusion proteins

Transfected cells express fairly high levels of intact hTR-FcΔH(SEQ IDNO:4). BHK cells also expressed hTR-Fc and hTR-FcΔCH₁ (SEQ ID NO:2) thatwere shown to have approximately the expected molecular mass. Thetransfection of baby hamster kidney (BHK) cells was performed by platingon 6-well tissue culture plates (Fisher Scientific, Pittsburgh, Pa.) orLab-Tek chamber slides (Nunc, Napierville, Ill.) coated withpoly-ornithine (Sigma, St. Louis, Mo.). Cells were transfected using thecalcium phosphate-based Stable Transfection Kit (Stratagene, San Diego,Calif.), using 1.0 mg DNA per ml of media per well. Cells weretransfected for 6-8 hours and grown for two days in Dulbecco's ModifiedEssential Medium (DMEM) with 10% heat inactivated fetal bovine serum(FBS). Polyclonal and monoclonal stable cell lines were selected usingG418 (Gibco-BRL, Gaithersburg, Md.) at a concentration of 1.0 mg/ml andmaintained at 0.25 mg/ml.

Expression and localization of reverse Fc by Western blot,immunostaining, and ELISA

Western blot analysis was used to determine relative hTR-Fc expression.Transfected cells were lysed directly in hot Laemmli sample buffer. Thelysates were resolved by SDS-PAGE and immunoblotted using goatanti-human IgG Fc specific HRP (horseradish peroxidase)-conjugatedantibody diluted 1:5000 (Sigma, St. Louis MO). Specific bands werevisualized by chemiluminescence (Pierce, Rockford, Ill.).

To determine if hTR-FcΔH (SEQ ID NO:4) targeted to the plasma membranein the desired orientation, surface expression of reverse human IgG Fcwas monitored by both immunostaining and ELISA. For immunofluorescencestaining, cells were processed essentially as described by Richards et.al. (J Cell Biol, 134:1157-68, 1996). Chimeric hTR-Fc molecules werevisualized using goat anti-human IgG Fc specific Cγ3 (carbocyanine)linked antibody (Jackson lmmunoResearch, West Grove, Pa.) diluted at1:1000. Nuclei were visualized by DAPI staining.

A stable BHK cell line expressing hTR-FcΔH (SEQ ID NO:4) produced highlevels of the reversed Fc, while wild-type cells exhibited no observablesignal. Similar membrane targeting was seen with the CH₁-deletion mutant(hTR-FcΔCH₁; SEQ ID NO:2), whereas intact IgG Fc with only Fab deletion(hTR-Fc) targeted to the endoplasmic reticulum almost exclusively.

Surface expression of reverse Fc was semi-quantitated by ELISA. ForELISA, cells were seeded at 100,000/well in a 96-well tissue cultureplate, as described by Margulies (In: Current Protocols in Immunology,Vol. 1, 1st Edition, Coligan et al., eds., John Wiley & Sons, Inc, 1994,p. 2.1.13). Goat anti-human IgG Fc alkaline phosphatase conjugate(Sigma, St. Louis Mo.) was added at 1:1000 dilution. Substrate PNPP wasadded and the plate was read after 30 minutes at OD₄₀₅ nm on a ThermoMaxPlate reader (Molecular Devices, Menlo Park, Calif.).

Cell surface-expressed human IgG₁ Fc from cells which showed positivesurface immunofluorescence was monitored by ELISA. Transfected cellsexhibited a very high signal versus wild-type BHK cells, which showed anear background signal (FIG. 2). These results confirm that thetransformed hTR-FcΔH (SEQ ID NO:4) cells express high levels of human Fcon the cell surface, consistent with the immunofluorescent data.

Characterization of cell surface expressed reverse Fc fusion proteins

To prove the design, immune complexes were created using BHK cells andanti-BHK antibodies. The effect of opsonized BHK cells on superoxideproduction by mouse macrophages were examined and the results arepresented in FIG. 3A. Preparation of mouse macrophages was performed asfollows: Macrophages were elicited by thioglycollate broth in nude mice(N:NIH(s)-nu/nuDF (Taconic Farms, Germantown, N.Y.). Mouse peritonealmacrophages were collected 72 hours after thioglycollate broth injectionand resuspended into 5×10⁶ cells/ml in RPMI+10% FCS. Fifty ml per wellof cells were seeded in a 96-well tissue culture plate and the plate wasincubated at 37° C. for at least 3 hours. The non-adherent cells wereremoved by washing three time with HBSS and 50 ml/well of HBSS wereadded.

BHK cells opsonized with different concentrations of antibodies produceda dose-dependent increase in superoxide production, while control cellsalone had no effect.

Various hTR-Fc fusion proteins (hTR-Fc, hTR-FcΔCH₁ [SEQ ID NO:2] andhTR-FcΔH [SEQ ID NO:4]) were expressed transiently or stably in BHKcells. The stable clones were derived from hTR-FcΔCH₁ [SEQ ID NO:1]andhTR-FcΔH [SEQ ID NO:3] transfected polyclonal cells, and the effect ofthese clones on superoxide production by mouse macrophages are shown inFIG. 3B. All subclones induced superoxide production. hTR-Fc did notexpress cell surface Fc therefore was eliminated from the study.BHK-FcΔH clone 3 was chosen for further characterization.

Functional analysis of BHK-FcΔH (3)

To assess whether cell surface expression of reverse Fc was biologicallyactive in terms of Fc receptor binding and complement fixation, Fcreceptor-mediated superoxide production in the human monocyte-like cellline was examined. Superoxide was measured spectrophotometrically as afunction of the cell's ability to reduce cytochrome c. Superoxideproduction was determined using the 96-well microtiter plate assay ofTao et al. (J Leukoc Biol, 58: 203-8, 1995). 25,000 cells/well of eitherBHK-WT (control), opsonized BHK cells, or BHK-reverse Fc clones wereadded to the assay plate containing either human monocyte-like U937cells (ATCC, Rockville, Md.) pre treated with recombinant humaninterferon-γ (R&D Systems, Minneapolis, Minn.) or lavaged mouseperitoneal macrophages. The O₂-release was measured as the superoxidedismutase (SOD) inhibitable reduction of cytochrome c at 550 nm by usinga ThermoMax Plate reader (Molecular Devices, Menlo Park, Calif.). Therate of O₂-production was monitored from time 0 to 60 mm afterstimulation.

BHK-FcΔH(3) cells induced an increase in superoxide production, whilewild-type cells had no effect.

Addition of FcγRI receptor specific antibody almost completely blockedthis induction (FIG. 4), indicating that the observed macrophageactivation was specifically mediated by FcγRl. Furthermore, superoxideproduction induced by BHK-FcΔH is dose-dependent. [Pre-incubation ofU937 cells with anti-CD64 F(ab′)2, a monoclonal antibody specific forhuman FcγRl, almost completely abolished BHK-FcΔH induced superoxideproduction (FIG. 4).]

The ability of cells expressing reverse Fc to activate complement

The ability of cells expressing reverse Fc to activate complement wasalso examined. In a C3a enzyme immunoassays, BHK-FcΔH cells failed toactivate complement, although immune complexes (IC) containing intactIgG activated complement effectively (FIG. 5). For the C3a enzymeimmunoassay, the effect of BHK-WT and BHK-IgGΔH on complement activationwas evaluated using QUIDEL C3a Enzyme Immunoassay kit (QUIDEL, SanDiego, Calif.). Human serum opsonized zymosan (immune complexes) wasused as a positive control. All specimen handling was carried outaccording to the guidelines provided by the manufacture. Human serum(complement) was incubated with BHK-WT, BHK-IgGΔH or immune complexesfor 1 hour at 37° C. Then serum samples were collected and subject toC3a-desArg quantitation. All calculations were made from a standardcurve provided by the manufacturer.

Other C3a enzyme immunoassays can be performed as described by Burger etal. (J Immunol, 141: 553-8, 1988); Mollnes et al. (Olin Exp Immunol, 73:484-8, 1988); and Hugh (In Laboratory and Research Methods in biologyand Medicine, Nakamura et al., Eds., New York, 1988, p. 443).

Complement Dependent Cytotoxicity.

BHK wild type (BHK-WT) and BHK-FcΔH (SEQ ID NO:4) cells were used astarget cells in an ADCC assay. All testing samples were set up in twogroups of triplicates and assayed employing a standard NationalInstitutes of Health (NIH) tissue typing technique (American Society forHistocompatibility and Immunogenetics (ASHI) Manual, 1994). One group oftriplicates was analyzed with sheep hypersensitized serum and BHK-WTcells, while the second group of triplicates was assayed in the samemanner with the addition of exogenously prescreened rabbit complement.In the case of BHK-FcΔH, the assay was performed in the absence orpresence of exogenous prescreened rabbit complement. Samples wereprepared using a two-color immunofluorescent microcytotoxic analysisprocedure. An one-hour incubation of 1 ml sheep serum and 1 mlcontaining 1,000 BHK cells was followed by a second one-hour incubationwith or without additional 5 ml rabbit complement. In the samemicrotiter well, the percentage of living cells (negative reactivity)was visualized using fluorescein diacetate, while the percentage of deadcells (positive reactivity) was visualized using propidium iodide. Allserum specimens were set up in 1:2 serial dilution to a maximum of a1:100,000 dilution. All data was scored using a Nikon Diaphot™ invertedfluorescence microscope with a 488 nm wavelength excitation.

In an ADCC assay containing BHK-WT or BHK-FcΔH (SEQ ID NO:4), no celllysis was observed in the presence of complement (TABLE 1), whereasanti-BHK sensitized serum (containing intact IgG) effectively lysed BHKtarget cells . These results suggest that although a high level of Fcwas expressed on the cell surface, the chimera of IgG₁ heavy chain withCH₁ deletion or CH₁ and hinge deletion did not activate complement,despite being a potent stimulus for macrophages.

TABLE 1 Cell Surface Fc CDC + Cell Type (OD ₄₀₅) complement BHK-WT 0.291± 0.052 BHK-FcΔH 2.803 ± 0.283

These results suggest that Fc expressed on the cell surface in areversed orientation can activate macrophages, and the observedactivation is mediated specifically through Fc receptors. The IgG₁ heavychain with CH₁ deletion (SEQ ID NO:2) or CH₁-hinge deletion (SEQ IDNO:4) gave similar levels of macrophage activation. The extracellularregion of the transferrin receptor used to anchor Fc (residues 89-97) isable to substitute for native hinge. The hTR fragment used in this studyis approximately equal in length, if not amino acid identity, to nativeIgG₁ hinge, and may effectively provide functions similar to that of thehinge region. Another critical function of hinge is to provideintermolecular disulfide bonds between heavy and light chains usingcritical cysteine residues. The hTR (1-97), which contains at least onecysteine (C89) necessary for TR dimerization, is able to mimic hinge byallowing multimer association of the hTR-FcΔH monomers.

In summary, a novel human IgG₁ Fc chimera has been constructed and thechimeric polypeptide was successfully expressed on the cell surface, ina reversed orientation. The Fc receptor binding property was retained inthe molecule, IgG₁ Fc chimera the complement activation capability wasabsent.

EXAMPLE 2 ENCAPSULATION PROCEDURE

Transformed cells from EXAMPLE 1 are encapsulated for transplantation ina host. The encapsulated cell devices typically include:

1) a semipermeable polyether sulfone (PES) hollow fiber membranefabricated by AKZO Nobel Faser AG;

2) a hub membrane segment;

3) a light cured methacrylate (LCM) resin leading end; and

4) a silicone tether.

The morphology of the device is as follows: The inner surface has apermselective skin. The wall has an open cell foam structure. The outersurface has an open structure, with pores up to 1.5 μm occupying 30±5%of the outer surface. Hollow fibers are fabricated from PES with anoutside diameter of 720 mm and a wall thickness of 100 mm (AKZO-NobelWippertal, Germany). These fibers are described in U.S. Pat. Nos.4,976,859 and 4,968,733. A PES#5 membrane, which has a MWGO of about 280kilodalton (kDa), is sometimes used. A PES#8 membrane, which has a MWGOof about 90 kDa, is at other times used. The semipermeable PES membranestypically have the following characteristics:

Internal Diameter 500 ± 30 μm Wall Thickness 100 ± 15 μm Force at Break100 ± 15 cN Elongation at Break  44 ± 10% Hydraulic Permeability  63 ± 8(ml/mn m² min Hg) nMWGO (dextrans) 280 ± 20 kDa

The components of the device are commercially available. The LCM glue isavailable from Ablestik Laboratories (Newark, Del.); Luxtrak AdhesivesLCM23 and LGM24). The tether material is available from SpecialtySilicone Fabricators (Robles, Ga.). The tether dimensions are 0.79 mmOD×0.43 mm ID×length 202 mm.

The encapsulation procedure is as follows: Fiber material is first cutinto 5 cm long segments and the distal extremity of each segment sealedwith a photopolymerized acrylic glue (LCM-25, ICI). Followingsterilization with ethylene oxide and outgassing, the fiber segments areloaded with a suspension of about 2×10⁵ transfected cells in a collagensolution (Zyderm® soluble bovine collagen) by a Hamilton syringe and a25 gauge needle through an attached injection port. The proximal end ofthe capsule was sealed with the same acrylic glue. Sometimes, thecollagen matrix was Zyplast®. The volume of the device for human use isapproximately 15-18 μl.

A silicone tether (Specialty Silicone Fabrication, Taunton, Mass.) (ID:690 μm; OD: 1.25 mm) is placed over the proximal end of the fiberallowing easy manipulation and retrieval of the device.

The foregoing description has been presented only for the purposes ofillustration and is not intended to limit the invention to the preciseform disclosed, but by the claims appended hereto.

14 1 1019 DNA Homo sapiens gene ()..) Description of SequenceRecombinant Polynucleotide 1 cccgaattcg ccacc atg atg gat caa gct agatca gca ttc tct aac ttg 51 Met Met Asp Gln Ala Arg Ser Ala Phe Ser AsnLeu 1 5 10 ttt ggt gga gaa cca ttg tca tat acc cgg ttc agc ctg gct cggcaa 99 Phe Gly Gly Glu Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln15 20 25 gta gat ggc gat aac agt cat gtg gag atg aaa ctt gct gta gat gaa147 Val Asp Gly Asp Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu 3035 40 gaa gaa aat gct gac aat aac aca aag gcc aat gtc aca aaa cca aaa195 Glu Glu Asn Ala Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys 4550 55 60 agg tgt agt gga agt atc tgc tat ggg act att gct gtg atc gtc ttt243 Arg Cys Ser Gly Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe 6570 75 ttc ttg att gga ttt atg att ggc tac ttg ggc tat tgt aaa ggg gta291 Phe Leu Ile Gly Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val 8085 90 gaa cca aaa act gag gga tcc gag ccc aaa tct tgt gac aaa act cac339 Glu Pro Lys Thr Glu Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His 95100 105 aca tgc cca ccg tgc cca gca cct gaa ctc ctg ggg gga ccg tca gtc387 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 110115 120 ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc cgg acc435 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 125130 135 140 cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac cctgag 483 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu145 150 155 gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat gccaag 531 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys160 165 170 aca aag ccg cgg gag gag cag tac aac agc acg tac cgt gtg gtcagc 579 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser175 180 185 gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag tacaag 627 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys190 195 200 tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa accatc 675 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile205 210 215 220 tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac accctg ccc 723 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr LeuPro 225 230 235 cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acctgc ctg 771 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr CysLeu 240 245 250 gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gagagc aat 819 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu SerAsn 255 260 265 ggg cag ccg gag aac aac tac aag acc acg cct ccc gtg ctggac tcc 867 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu AspSer 270 275 280 gac ggc tcc ttc ttc ctc tac agc aag ctc acc gtg gac aagagc agg 915 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys SerArg 285 290 295 300 tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg catgag gct ctg 963 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His GluAla Leu 305 310 315 cac aac cac tac acg cag aag agc ctc tcc ctg tct ccgggt aaa 1008 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys320 325 330 tgaagcttgg g 1019 2 331 PRT Homo sapiens 2 Met Met Asp GlnAla Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu 1 5 10 15 Pro Leu SerTyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 25 30 Asn Ser HisVal Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala 35 40 45 Asp Asn AsnThr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly 50 55 60 Ser Ile CysTyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly 65 70 75 80 Phe MetIle Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 85 90 95 Glu GlySer Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 100 105 110 CysPro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 115 120 125Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 130 135140 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 145150 155 160 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys ProArg 165 170 175 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val LeuThr Val 180 185 190 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys CysLys Val Ser 195 200 205 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr IleSer Lys Ala Lys 210 215 220 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr LeuPro Pro Ser Arg Asp 225 230 235 240 Glu Leu Thr Lys Asn Gln Val Ser LeuThr Cys Leu Val Lys Gly Phe 245 250 255 Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu 260 265 270 Asn Asn Tyr Lys Thr Thr ProPro Val Leu Asp Ser Asp Gly Ser Phe 275 280 285 Phe Leu Tyr Ser Lys LeuThr Val Asp Lys Ser Arg Trp Gln Gln Gly 290 295 300 Asn Val Phe Ser CysSer Val Met His Glu Ala Leu His Asn His Tyr 305 310 315 320 Thr Gln LysSer Leu Ser Leu Ser Pro Gly Lys 325 330 3 974 DNA Homo sapiensDescription of Sequence Recombinant Polynucleotide 3 cccgaattcg ccaccatg atg gat caa gct aga tca gca ttc tct aac ttg 51 Met Met Asp Gln AlaArg Ser Ala Phe Ser Asn Leu 1 5 10 ttt ggt gga gaa cca ttg tca tat acccgg ttc agc ctg gct cgg caa 99 Phe Gly Gly Glu Pro Leu Ser Tyr Thr ArgPhe Ser Leu Ala Arg Gln 15 20 25 gta gat ggc gat aac agt cat gtg gag atgaaa ctt gct gta gat gaa 147 Val Asp Gly Asp Asn Ser His Val Glu Met LysLeu Ala Val Asp Glu 30 35 40 gaa gaa aat gct gac aat aac aca aag gcc aatgtc aca aaa cca aaa 195 Glu Glu Asn Ala Asp Asn Asn Thr Lys Ala Asn ValThr Lys Pro Lys 45 50 55 60 agg tgt agt gga agt atc tgc tat ggg act attgct gtg atc gtc ttt 243 Arg Cys Ser Gly Ser Ile Cys Tyr Gly Thr Ile AlaVal Ile Val Phe 65 70 75 ttc ttg att gga ttt atg att ggc tac ttg ggc tattgt aaa ggg gta 291 Phe Leu Ile Gly Phe Met Ile Gly Tyr Leu Gly Tyr CysLys Gly Val 80 85 90 gaa cca aaa act gag gga tcc gca cct gaa ctc ctg ggggga ccg tca 339 Glu Pro Lys Thr Glu Gly Ser Ala Pro Glu Leu Leu Gly GlyPro Ser 95 100 105 gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atgatc tcc cgg 387 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met IleSer Arg 110 115 120 acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cacgaa gac cct 435 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His GluAsp Pro 125 130 135 140 gag gtc aag ttc aac tgg tac gtg gac ggc gtg gaggtg cat aat gcc 483 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu ValHis Asn Ala 145 150 155 aag aca aag ccg cgg gag gag cag tac aac agc acgtac cgt gtg gtc 531 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val 160 165 170 agc gtc ctc acc gtc ctg cac cag gac tgg ctg aatggc aag gag tac 579 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn GlyLys Glu Tyr 175 180 185 aag tgc aag gtc tcc aac aaa gcc ctc cca gcc cccatc gag aaa acc 627 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr 190 195 200 atc tcc aaa gcc aaa ggg cag ccc cga gaa cca caggtg tac acc ctg 675 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln ValTyr Thr Leu 205 210 215 220 ccc cca tcc cgg gat gag ctg acc aag aac caggtc agc ctg acc tgc 723 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln ValSer Leu Thr Cys 225 230 235 ctg gtc aaa ggc ttc tat ccc agc gac atc gccgtg gag tgg gag agc 771 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu Trp Glu Ser 240 245 250 aat ggg cag ccg gag aac aac tac aag acc acgcct ccc gtg ctg gac 819 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProPro Val Leu Asp 255 260 265 tcc gac ggc tcc ttc ttc ctc tac agc aag ctcacc gtg gac aag agc 867 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu ThrVal Asp Lys Ser 270 275 280 agg tgg cag cag ggg aac gtc ttc tca tgc tccgtg atg cat gag gct 915 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser ValMet His Glu Ala 285 290 295 300 ctg cac aac cac tac acg cag aag agc ctctcc ctg tct ccg ggt aaa 963 Leu His Asn His Tyr Thr Gln Lys Ser Leu SerLeu Ser Pro Gly Lys 305 310 315 tgaagcttgg g 974 4 316 PRT Homo sapiens4 Met Met Asp Gln Ala Arg Ser Ala Phe Ser Asn Leu Phe Gly Gly Glu 1 5 1015 Pro Leu Ser Tyr Thr Arg Phe Ser Leu Ala Arg Gln Val Asp Gly Asp 20 2530 Asn Ser His Val Glu Met Lys Leu Ala Val Asp Glu Glu Glu Asn Ala 35 4045 Asp Asn Asn Thr Lys Ala Asn Val Thr Lys Pro Lys Arg Cys Ser Gly 50 5560 Ser Ile Cys Tyr Gly Thr Ile Ala Val Ile Val Phe Phe Leu Ile Gly 65 7075 80 Phe Met Ile Gly Tyr Leu Gly Tyr Cys Lys Gly Val Glu Pro Lys Thr 8590 95 Glu Gly Ser Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe100 105 110 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro GluVal 115 120 125 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu ValLys Phe 130 135 140 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala LysThr Lys Pro 145 150 155 160 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg ValVal Ser Val Leu Thr 165 170 175 Val Leu His Gln Asp Trp Leu Asn Gly LysGlu Tyr Lys Cys Lys Val 180 185 190 Ser Asn Lys Ala Leu Pro Ala Pro IleGlu Lys Thr Ile Ser Lys Ala 195 200 205 Lys Gly Gln Pro Arg Glu Pro GlnVal Tyr Thr Leu Pro Pro Ser Arg 210 215 220 Asp Glu Leu Thr Lys Asn GlnVal Ser Leu Thr Cys Leu Val Lys Gly 225 230 235 240 Phe Tyr Pro Ser AspIle Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 245 250 255 Glu Asn Asn TyrLys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 260 265 270 Phe Phe LeuTyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 275 280 285 Gly AsnVal Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 290 295 300 TyrThr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 305 310 315 5 33 DNAArtificial Sequence Description of Artificial Sequence Primer 5cccggatccg cctccaccaa gggcccatgc gtc 33 6 33 DNA Artificial SequenceDescription of Artificial Sequence Primer 6 cccaagcttc atttacccggagacagggag agg 33 7 32 DNA Artificial Sequence Description of ArtificialSequence Primer 7 cccggatccg ccaccatgat ggatcaagct ag 32 8 31 DNAArtificial Sequence Description of Artificial Sequence Primer 8cccgaattcg ccaaaacgac acccccatct g 31 9 32 DNA Artificial SequenceDescription of Artificial Sequence Primer 9 cccaagcttc atttaccaggagagtgggag ag 32 10 32 DNA Artificial Sequence Description of ArtificialSequence Primer 10 cccgaattcg ccaccatgat ggatcaagct ag 32 11 30 DNAArtificial Sequence Description of Artificial Sequence Primer 11tgtccttttg gcctcagttt ttggttctac 30 12 30 DNA Artificial SequenceDescription of Artificial Sequence Primer 12 ccaaaaactg aggccaaaacgacaccccca 30 13 32 DNA Artificial Sequence Description of ArtificialSequence Primer 13 cccgaattcg ccaccatgat ggatcaagct ag 32 14 30 DNAArtificial Sequence Description of Artificial Sequence Primer 14gtgtgcacac cgctggacag ggatccagag 30

What is claimed is:
 1. A cell transformed in vitro comprising a vector,said vector comprising a promoter operatively linked to a polynucleotidesequence encoding a fusion protein comprising the Fc portion of an IgGmolecule linked at the amino terminus to the transferrin receptor hingeregion, wherein upon expression, the fusion protein is expressed on thecell surface.
 2. The cell according to claim 1, wherein the cell is ahuman cell.
 3. The cell according to claim 1, wherein the cell is arodent cell.
 4. The cell according to claim 3, wherein the rodent cellis a mouse cell.
 5. A vector comprising a promoter operatively linked toa polynucleotide sequence encoding a fusion protein comprising the Fcportion of an IgG molecule linked at the amino terminus to thetransferrin receptor hinge region.
 6. A composition comprising: (a) aplurality of cells according to claim 1; and (b) a jacket surroundingthe plurality of cells, wherein the jacket comprises a permselectivemembrane.